Cystic fibrosis (CF) is the most common lethal genetic disease affecting Caucasian populations. The illness is caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that serves a critical role maintaining lung health and homeostasis. Normal CFTR gating or "activation" requires phosphorylation of the regulatory domain (RD) and ATP-driven dimerization of the two nucleotide binding domains (NBDs). CFTR mutations may engender one of a number of defects that reduce chloride transport, including reduced expression, decreased presence at the membrane, and insufficient gating. 'Potentiator'compounds capable of improving the gating of mutant CFTR have been discovered by high-throughput drug screening programs, and the first of these compounds, VX-770, has recently shown considerable promise as part of Phase I testing in CF subjects. However, the mechanism(s) by which CFTR potentiators open the mutant chloride channel are not known. The first Aim of this study encompasses a detailed analysis of the mechanisms underlying several CFTR potentiator compounds. Biochemical study of isolated RD, electrophysiologic analysis of full-length CFTR, and enzymatic studies will determine effects on the cAMP-based RD phosphorylation cascade. Calorimetric (thermal melting) experiments will evaluate potentiator binding to individual NBDs. In the second Aim, we will investigate the relevance of these findings to in vivo potentiator activation of CFTR in mice. This will include both AF508-CFTR, the most common disease-causing CFTR mutation, and less frequent mutations, e.g. G551D and R177H. Potentiators of CFTR present an opportunity to better understand the basic means underlying activation of this important ion channel, and the domain interactions utilized by CFTR and other ATP binding cassette gene family members. An understanding of potentiator mechanism of action is also required for a more rational and efficient approach to optimizing compounds such as these for human use. PUBLIC HEALTH RELEVANCE: Cystic Fibrosis is a common genetic disease in the U.S. In this project we aim to better define the types of drugs that will help alleviate the disease by correcting defective function of the epithelial ion channel.